ASTM E 2149-10

Adapted Test Protocol

Preparation of Bacteria Culture

  1. Transfer 100 µL of bacteria from glycerol stock into 3 mL compatible liquid medium in a 15 mL test tube. Cultivate culture overnight at 200 rpm and 37 °C.
  2. Next day, inoculate 500 µL aliquot of preculture into 100 mL of a compatible medium in a 500 mL Erlenmeyer and incubate at 37 °C with 200 rpm shaking until OD600 value reaches 0.1. Prepare 9 Erlenmeyer to have 3 replicates of sample with antibacterial agent, 3 replicates of samples without antibacterial agent and 3 replicates of culture without sample.

 

Preparation of Samples

  1. Cut polymer samples into square pieces of 1 cm2 (30 pieces).
  2. Prepare samples of 1 cm2 without antibacterial agent treatment as control group (30 pieces).
  3. Remove impurities (e.g. wash with acetone if applicable).
  4. Sterilize samples (e.g. autoclave at 121 °C for 20 minutes if applicable).

 

Monitoring Bacterial Lag-Log Phases in the Presence of Antibacterial Agent

  1. When OD600 values of bacteria reaches 0.1, add 10 pieces of specimen with and without antibacterial treatment into 500 mL Erlenmeyer and continue shaking at 200 rpm at 37 C°.
  2. Measure OD600 value of bacteria in every 30 minutes until it reaches 2.0.
  3. Standardize relative OD600 values to evaluate influence of antibacterial polymer on the growth rate of bacteria.
  4. Use means of 3 cultures to create lag-log phase graphs.

 

Media Preferences

  1. Luria-Bertani for E. coli DH5α.
  2. Mueller-Hinton II for S. typhimurium.
  3. High Salt Medium for B. subtilis BCC 6327.
    • 665 mL of distilled water.
    • 100 mL of 10X Spizizen’s salt.
    • 25 mL of 20% w/v glucose.
    • 50 mL of 0.1% w/v L-tryptophan.
    • 10 mL of 2% w/v casein.
    • 50 mL of 10% w/v yeast extract.
    • 100 mL of 8% w/v arginine.
    • 4% of w/v of histidine.

 

Reference: J. APPL. POLYM. SCI 2016. DOI: 10.1002/APP.43331

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